Influence of Glucose on the Transmembrane Action Potential of Anoxic Papillary Muscle

The response of the cat papillary muscle to anoxia has been found to alter depending on the glucose concentration in the medium. At a glucose concentration of 5 mM anoxia caused a marked reduction in force of contraction and action potential duration within 20 minutes. At a glucose concentration of 50 mM anoxia induced similar changes in the force of contraction but little or no change in action potential duration. Elevation of glucose concentration during an anoxic interval reversed the anoxia-induced changes in action potential but had little effect on force of contraction. This effect of glucose could be partially duplicated by xylose and 2-deoxyglucose and in addition, 2-deoxyglucose has been found to prevent the effect of subsequently added glucose. These sugars appear to be transported by a system responsible for glucose transport but are not metabolized to any extent. It would appear therefore that transport of glucose is in some way related to transport of potassium as increased potassium permeability is thought by many to be responsible for anoxia-induced changes in action potential duration.     

Detection of motor unit action potentials with surface electrodes: influence of electrode size and spacing

A model of the motor unit action potential was developed to investigate the amplitude and frequency spectrum contributions of motor units, located at various depths within muscle, to the surface detected electromyographic (EMG) signal. A dipole representation of the transmembrane current in a three-dimensional muscle volume was used to estimate detected individual muscle fiber action potentials. The effects of anisotropic muscle conductance, innervation zone location, propagation velocity, fiber length, electrode area, and electrode configuration were included in the fiber action potential model. A motor unit action potential was assumed to be the sum of the individual muscle fiber action potentials. A computational procedure, based on the notion of isopotential layers, was developed which substantially reduced the calculation time required to estimate motor unit action potentials. The simulations indicated that: 1) only those motor units with muscle fibers located within 10–12 mm of the electrodes would contribute significant signal energy to the surface EMG, 2) variation in surface area of electrodes has little effect on the detection depth of motor unit action potentials, 3) increased interelectrode spacing moderately increases detection depth, and 4) the frequency content of action potentials decreases steeply with increased electrode-motor unit territory distance.     

Proteomic analysis of apoptosis induction in human lung cancer cells by recombinant MVL

Lung cancer is still difficult to treat by current chemotherapeutic procedures. We recently found that MVL, an anti-HIV lectin from blue-green algae Microcystis viridis, also has antitumor activity. The objective of this study was to investigate apoptosis-inducing activity of recombinant MVL (R-MVL) and proteomic changes in A549 cells, and to identify the molecular pathways responsible for the anti-cancer action of R-MVL. We found that R-MVL induces A549 cells apoptosis in a dose-dependent manner by using MTT assay, fluorescent microscope (FM) and flow cytometry (FCM), and the IC50 was calculated to be 24.12 μg/ml. Subsequently, 7 altered proteins in R-MVL-treated A549 cells were identified, including upregulated aldehyde dehydrogenase 1 and β-actin, and five downregulated proteins: heat shock protein 90, heat shock 60, plastin 3, tropomyosin 3, and β-tubulin. Further bioinformatics analysis predicted the potential pathways for R-MVL to induce apoptosis of A549 cells. In conclusion, this is the first report to investigate anti-cancer activity of R-MVL and its mechanism of action by proteomics analysis. Our observations provide potential therapeutic targets for lung cancer inhibitor intervention and implicated the development of novel anti-cancer therapeutic strategies.     

Neuromuscular modulation in Limulus by both octopamine and proctolin

Both octopamine and proctolin potentiate nerve-evoked skeletal muscle contractions in the horseshoe crab, Limulus. The threshold concentration for octopamine was 10^?9 to 10^?8M, while for proctolin it was 3 × 10^?9M. Norepinephrine and dopamine produced effects similar to octopamine but at higher thresholds; tyramine and serotonin were ineffective. Octopamine caused significant increases in amplitudes of excitatory postsynaptic potentials (epsps) of muscle fibers, but had little effect on muscle fiber input resistance or membrane potential. Also, octopamine did not affect depolarization of muscle fibers and subsequent contraction due to the direct action of exogenously applied glutamate. These results suggest that octopamine potentiates nerve-evoked contractions primarily by facilitating release of neuromuscular transmitter. At concentrations above 10^?7M, however, octopamine sometimes caused muscle spikes in response to motoneuron stimulation, a finding that suggests that octopamine may also have some postsynaptic action. Proctolin potentiated the muscle contractions evoked by glutamate but had little effect on glutamate-evoked muscle fiber depolarization, muscle fiber input resistance, or membrane potential. Thus, proctolin appears to act directly on skeletal muscle to enhance contractility. The proctolin-induced potentiations of contraction were sometimes accompanied by modest increases in epsp amplitude, so that unlike lobster skeletal and Limulus cardiac neuromuscular preparations, proctolin may have a secondary direct synaptic effect. Both octopamine and proctolin have been found in Limulus cardiac ganglion. This potential access to the hemolymph and the relatively low threshold concentrations needed for physiological action suggest that octopamine and proctolin could function as hormonal modulators of neuromuscular function in Limulus.     

Length-dependent deactivation of ventricular trabeculae in the bivalve, Spisula solidissima

Shortening-deactivation has been identified and characterized in ventricular trabeculae of the bivalve, Spisula solidissima (Heterodonta, Mactridae). This muscle had ultrastructural similarities to vertebrate smooth muscle. Deactivation was defined as the fraction of maximal force lost during a contraction when a muscle is shortened rapidly (by a quick-release, QR) to a known length, relative to a control isometric contraction at that same length. The magnitude of deactivation was dependent on the size of the release and the point at which the release was applied during the cycle of contraction. QR/quick-stretch (QS) perturbations at the same point during the contraction resulted in negligible deactivation. The magnitude of deactivation was independent of shortening rate. Deactivation was attenuated by applying caffeine (100 μM) and blocked with high extracellular Ca²⁺ (56 mM). The Ca²⁺ ionophore, A23187 (10 μM), augmented deactivation as did the positive inotrope serotonin (100 nM). Treatment with ryanodine (5 μM) had no significant effect on deactivation. These results suggest that a reduction in Ca²⁺ at the contractile element and/or sequestration of Ca²⁺ may occur during shortening. Deactivation may minimize the magnitude of work done during active shortening of bivalve cardiac muscle, particularly against the low afterload exhibited in the bivalve peripheral circulatory system. Intracellular Ca²⁺ fluxes during sudden length perturbations may explain the effect of stretch on action potential duration in the bivalve heart, as shown previously.     

Killing of bacteria with electric pulses of high field strength

Summary Bacteria of the typeE. coli K12 have been treated in experiments using high-voltage pulses of short time (µs) as a killing agent. The role of different experimental parameters has been studied: kind of electrolyte, concentration, length of pulses, field strength, pH and temperature. Electrolytes with bivalent cations were found to reduce the lethal action. The relative rate of killed bacteria was shown to be mainly governed by the field strength and the treatment time, which is defined by the product of pulse number and decay time constant. From the obtained results a function has been developed which enables the precalculation of the killing rate forE. coli, provided that certain limits of experimental conditions are considered. No correlation between the applied electric energy and the lethal effect could be found.     

Extracellular potentials of a single myelinated nerve fiber in an unbounded volume conductor

The extracellular potentials of a single myelinated nerve fiber in an unbounded volume conductor were studied. The spatial distribution of the transmembrane potential was obtained by integrating the system of partial differential equations characterizing the electric processes in the active myelinated nerve fiber. The spatial distribution of the extracellular potentials at various radial distances in the volume conductor were calculated using the line source model. Up to a certain radial distance (500 m) the discontinuity of the action potential propagation is reflected in the extracellular potentials, while further in the volume conductor the potentials are smooth. The effect of the fiber diameter and the internodal distance on the volume conductor potentials as well as the changes in the magnitude of the extracellular potential (in the time domain) between two adjacent nodes at various radial distances were studied. The radial decline of the peak-to-peak amplitude of the extracellular potential depends on the radial coordinater of the field point and increases with the increase ofr.     

Investigation of the temperature dependence of the cross bridge parameters for attachment,force generation and detachment as deduced from mechano-chemical studies in glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus

Birefringence change during excitation was studied by using Nitellopsis obtusa. The velocity change of cytoplasmic streaming during an action potential was measured simultaneously by fluctuation analysis of transmitted light intensity. The origin of the retardation change was discussed by comparing optical retardation change to the time course of the action potential, the cytoplasmic streaming velocity change and the cell contraction.By the time course analysis of retardation change, we concluded that the change of the birefringence might be the sum of the changes of cytoplasmic flow and that of the size of length and diameter of the cell. But it is still difficult to separate the change to its components.     

A novel cottong ovule culture: Induction, growth, and characterization of submerged cotton fibers (Gossypium hirsutum L.)

Summary The growth of submerged cotton (Gossypium hirsutum L.) fibers from cultured ovules has been investigated. The results indicate that exogenous plant hormone levels regulate the induction of submerged fiber growth. The age of ovules at induction is also important. Cell diameter, wall thickness, and cell length of submerged fibers were measured and compared with air-grown fibers and fibers grown in vivo (produced by cotton plants grown in the greenhouse). Various cellwall thickening patterns were observed among submerged fibers, while only one predominant cell-wall deposition pattern was produced in air-grown fibers and in fibers produced in vivo. The diameter of submerged fibers was about the same as that of air-grown fibers but about 22% less than that of fibers grown, in vivo. It appears that the secondary cell wall thickenings are initiated earlier in submerged fibers. The cell-wall thickness of submerged fibers, at 41 d post anthesis (DPA), was 51% greater than that of fibers grown in vivo, whereas the cell-wall thickness of air-grown fibers was 42% less than that of fibers produced in vivo. The cell length of submerged fibers was approximately half that of fibers grown in vivo. and the air-grown fiber length was about two-thirds of fibers grown in vivo. The age of ovules at induction affects the outcome of the air-grown fiber-cell length, but does not appear to affect the length of submerged fiber cells. To produce submerged fiber growth, we found that the optimal age of ovules at induction was 0 DPA, and the optimal medium (with a GA₃ of 0.5 μM and an IAA range of 5-20 μM) depends on the time of ovule induction (−2 to+2DPA). We conclude that conditions leading to submerged cotton fiber growth have great potential for (a) direct monitoring of growth and making precise, detailed measurements during fiber growth and development; (b) producing cellulose and fibers in vitro more efficiently than earlier ovule-culture methods; and (c) using these unique cultures to obtain a better understanding of signal transduction and gene expression leading to growth, development, and programmed cell death in the life history of the cotton fiber.     

Vision in microalgae

Flagellate green algae such as Chlamydomonas and related genera are guided by their eyes to places where light conditions are optimal for photosynthetic growth. These eyes constitute the simplest and most common visual system found in nature. The eyes contain optics, photoreceptors and the elementary components of a signal-transduction chain. Rhodopsin serves as the photoreceptor, as it does in animal vision. Upon light stimulation, its all-trans-retinal chromophore isomerizes into 13-cis and activates a photoreceptor channel which leads to a rapid Ca²⁺ influx into the eyespot region. At low light levels, the depolarization activates small flagellar currents which induce in both flagella small but slightly different beating changes resulting in distinct directional changes. In continuous light, Ca²⁺ fluxes serve as the molecular basis for phototaxis. In response to flashes of higher energy the larger photoreceptor currents trigger a massive Ca²⁺ influx into the flagella which causes the well-known phobic response. The identification of proteins contributing to this signalling system has just begun with the isolation and cloning of the opsins from Chlamydomonas and Volvox. These plant opsins are highly charged, are not typical seven-helix receptors, and are believed to form a protein complex with the photoreceptor channel. In Spermatozopsis, a G-protein has been found which interacts either directly with the rhodopsin or with the rhodopsin-ion channel complex. By using insertional mutagenesis, genes coding for proteins that are involved in signalling have been tagged. One of them is connected to the flagellar channel and crucial for the flagellar action potential. Elucidation of photoreception in flagellated algae will provide deeper insight into the development of visual systems, starting from single-celled organisms and moving up through higher animals. Received: 10 March 1997 / Accepted: 18 April 1997     

Effects of quercetin and verapamil on membrane potential in the liverwort Conocephalum conicum

Quercetin is a very common flavonoid widely distributed in many plants. The flavonoid intake has been linked to the prevention of human diseases including cancer. Flavonoids possess also a broad spectrum of effects on plants. Quercetin is involved in Ca²⁺ transport and metabolism. The present study was designed to check the effects of quercetin alone and in combination with verapamil on the resting and action potentials in the liverwort Conocephalum conicum. The application of 59·10⁻⁶ mol·dm⁻³ quercetin caused an increase of action potential amplitudes. During the 3rd and 4th hour of treatment an increase by 20–22 % with respect to the control was observed. No changes were found in the resting potential in quercetin treated plants. Verapamil, a calcium channel inhibitor, caused gradual decrease of action potential amplitudes. Quercetin, when added together with verapamil, prevented its inhibitory effect. Interactions between quercetin and Ca²⁺ transport are discussed.     

Changes in geometry of activily shortening unipennate rat gastrocnemius muscle

Muscle geometry of the unipennate medial gastrocnemius (GM) muscle of the rat was examined with photographic techniques during isometric contractions at different muscle lengths. It was found that the length of fibers in different regions of GM differs significantly, and proximal aponeurosis length varies significantly from distal aponeurosis length; the angle of the aponeurosis with the muscular action differs significantly among regions at short muscle lengths (full contraction). These data support the idea that the unipennate GM cannot be represented by a parallelogram in a two-dimensional analysis. As the muscle shortens, the area of the mid-longitudinal plane of the GM decreases by 24%, a decrease that may be explained by assuming fiber diameter to increase in all directions. The angle between fiber and aponeurosis is determined by more than fiber length. Hence, such important assumptions as a parallelogram with constant area and fiber angle γ changes determined by fiber length changes, freqently used in the theoretical analysis of the morphological mechanism of unipennate muscle contraction, do not hold for the unipennate GM of the rat. Length of the sarcomere within the mid-longitudinal plane of GM varies from 1.92 to 2.14 μm among the different muscle regions at muscle optimum length (length at which force production is highest), whereas shortening to 6 mm less than optimum length produces a range of sarcomere lengths from 0.89 to 1.52 μm. These data suggest that fibers located in different regions of the GM reach their optimum and slack lengths at various muscle lengths. © 1993 Wiley-Liss, Inc.     

Evaluation of proximity inhibition of DNA synthesis in 3T3 cells.

A microphotometric technique that displays rapid length changes of Spirostomum has been used to follow the variation with temperature of three kinetic parameters of myonemal contraction: contraction rate, relaxation rate and stimulus duration at threshold. In each case the exponential form of the relationship indicated that the gross rate constant might be equated with the limiting rate constant, k, of a driving chemical reaction, and from standard expressions of chemical kinetics the change in activation free energy appropriate to this reaction has been computed. The thermal dependence of contraction is described by an activation enthalpy (ΔLH?) of 21.7 kcal mol^?1, and the activation entropy (ΔLS?) of 26.8 e.u. is consistent with a model of contraction requiring neutralization of fixed myonemal charges by divalent cations. The analysis of thermal dependence of relaxation gives a negative activation entropy, a result predicted for a rate-limiting reaction involving dissociation of a neutral molecule. On the other hand, values of ΔLS? and ΔLH? for relaxation fall close to an isokinetic correlation drawn in the literature from analysis of the thermal dependence of ciliary beat frequency in different organisms, and for which breakdown of an ATP-ATPase complex could be the common rate-limiting reaction. ΔLS? for stimulus duration suggests that the rate-limiting step in excitation-contraction coupling is a reaction between ions of like charge, or ion pair formation from a neutral molecule.     

Induction of DNA double-strand breaks by zeocin in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and the role of increased DNA double-strand breaks rejoining in the formation of an adaptive response

This study aimed to test the potential of the radiomimetic chemical zeocin to induce DNA double-strand breaks (DSB) and “adaptive response” (AR) in Chlamydomonas reinhardtii strain CW15 as a model system. The AR was measured as cell survival using a micro-colony assay, and by changes in rejoining of DSB DNA. The level of induced DSB was measured by constant field gel electrophoresis based on incorporation of cells into agarose blocks before cell lysis. This avoids the risk of accidental induction of DSB during the manipulation procedures. Our results showed that zeocin could induce DSB in C. reinhardtii strain CW15 in a linear dose-response fashion up to 100 μg ml⁻¹ which marked the beginning of a plateau. The level of DSB induced by 100 μg ml⁻¹ zeocin was similar to that induced by 250 Gy of gamma-ray irradiation. It was also found that, similar to gamma rays, zeocin could induce AR measured as DSB in C. reinhardtii CW15 and this AR involved acceleration of the rate of DSB rejoining, too. To our knowledge, this is the first demonstration that zeocin could induce AR in some low eukaryotes such as C. reinhardtii.     

Stiffness and tension during and after sudden length changes of glycerinated single insect fibrillar muscle fibres

Rapid length changes were applied (within 0.2 ms or 0.4 ms) to single isometrically contracted glycerol extracted muscle fibres of the dorsal longitudinal muscle ofLethocerus maximus suspended in an Ca²⁺ and ATP containing solution at 20–23‡ C. Force transients and the fibre stiffness were measured during and after rapid length changes. At length changesbelow 0.5% of the initial fibre length (∼ 2.4 Μm sarcomere length) the mechanical transients were characterized as follows: (1) After stretch and after release the force regains at least partly the value of tension before the length change within a quick phase of tension recovery. The quick phase induced by stretch was nearly completed within 1–2 ms. (2) A pulse in length of 1.5 ms duration, i.e., a stretch followed by a release to the initial length or a release followed by a stretch to the initial length, was applied to the fibre. The force transient induced by this procedure regains after the second length change the value of the isometric tension before the procedure. (3) The stiffness was constant during each length change of the “pulse” and was equal during the first and the second length changes. These findings are predicted by the muscle contraction model of Huxley and Simmons (1971): The identical force before and after a length pulse may indicate that the rotation of cross bridges after the first length change is followed by a rotation into the original position after the second length change. The constancy of the stiffness during the length changes may indicate a Hookean elastic element of the cross bridge. The similarity of the stiffness during the first and the second length changes, i.e., before and after the quick phase, gives evidence that the quick phases after stretch and after release are not accompanied by a change in the net number of attached cross bridges. If stretches ofmore than 0.5% of the initial length were applied, the mechanical transient of the muscle fibre changed as follows: (1) An ultra fast tension decay phase (duration < 0.4 ms) was observed in addition to the slower decay phase induced by the smaller stretches. (2) If the initial stretch was followed by a release to the initial length, no fast recovery phase was observed, which returns the force to the value before the stretch. The reduced tension value persists for a longer period in time than 10 ms. (3) If the muscle was stretched and released repetitively an ultra fast quick phase was induced only by the first stretch. (4) The stiffness increased during stretch, but was found to be the same in the isometrically contracting muscle and after the quick tension decay phase following a large stretch. These findings indicate that the contraction model of Huxley and Simmons has to be extended by a further process additional to cross bridge rotation in case of large stretches (> 0.5%L ini). The findings are taken to indicate a rapid detachment and reattachment of overstrained cross bridges, i.e., a cross bridge slippage induced by large stretches.     

Evaluation of maize productivity considering solar energy use limitation by environmental factors

Evaluation of maize productivity under different climatic conditions was made by determination of the amount of the incident solar radiation energy in the PAR range, which can be potentially used by plants for photosynthesis. An irradiance, which can be stored in primary photosynthesis, designated as photosynthetic energy, W _ph, was estimated taking into account the action spectra of photosynthesis. Limitation of the W _ph usage, owing to unfavorable environmental factors was considered. Quantitative evaluation of limitations by two such factors, air temperature and soil water potential, was made by means of the coefficients F(i), which were defined as the ratio between the photosynthetic rate at a given value of a particular environmental factor and that at the optimal value for this factor. The coefficients F(i), were determined from the dependencies of the photosynthesis rate on air temperature and soil water potential as obtained in chamber and field experiments. In general terms, the fraction of W _ph, which can be utilized under a given climatic condition, was named bioclimatic potential, W _pc. In our model, the effect of monodominancy, when strong action of one factor suppresses the influence of any other factor, was considered. In this case, the bioclimatic potential, designated W′_pc, was calculated by multiplying W _ph times the coefficient F, for the factor which was most limiting during the period of measurement. There was close correlation between values of bioclimatic potential for the period of vegetation, W′_pc,v, and total dry matter. W′_pc,v use efficiency in the maize crop was also evaluated for five variants of mineral nutrition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mean-square amplitudes of the longitudinal vibrational of helical polymers

A simple model for the longitudinal acoustical vibrations of helical polymers has been developed. This model consists of a linear string of masses, M, connected by springs. The mass must be taken as that of one helical turn of the true helix. It has been found that all of the previously observed or calculated frequencies for the longitudinal acoustical modes of helical polymers can be calculated from this model using only one adjustable parameter, the force constant f, between the turns, for each type of helix. Using this simplified model it has been possible to obtain the rms amplitudes of the changes in the overall end-to-end length of helical polymers as a function of their chain length and temperature. At room temperatures the rms end-to-end length is found to vary from a few tenths of an angstrom for hydrocarbon chains of the length found in phospholipid membrane bilayers to several angstroms in a rigid α-helix of 100 peptide residues. The damping of these α-helical modes is not considered but may be appreciable in solution.     

Organization and chromosomal localization ofβ-tubulin genes inLeishmania donovani

The genomic organization and chromosomal location of theβ-tubulin isogenes inLeishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned β-tubulin gene. We have cloned aβ-tubulin gene fragment, 3.3 kbp long, from genomic DNA ofLeishmania donovani using a heterologousβ-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11–15 copies of theβ-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3.5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of theβ-tubulin clone and is found to be nearly identical to that ofLeishmania mexicana amazonensis.     

Ionic transporting systems of skeletal muscle in relation with innervation and their involvement in myotonic diseases

Excitation-contraction coupling describes the series of events that begins with propagated action potential on the muscle fiber surface membrane and leads to the twitch contraction of the fiber. The generation of an action potential during excitation requires rapid sequential changes in membrane conductances of Na⁺, Ca²⁺, and K⁺ ions that depend upon the opening and closing of the respective channels. Myotonic disorders are inherited diseases whose clinical manifestations include electrophysiological signs such as increased excitability and delayed relaxation of the muscles after voluntary contraction. All these disorders appears to be due to an abnormality of the muscle itself since they persist after section or blocking of the motor nerve after curarization. Most experimental and clinical data suggest that human myotonia arises from genetically-induced structural and functional alterations of the muscle membrane. The purpose of this article is to focus on the more recent developments in the molecular and pharmacological analysis of cation transporting systems such as ionic channels and (Na⁺, K⁺) ATPase in myotonic disorders.Special issue dedicated to Dr. Lawrence Austin.     

Pea Fiber and Wheat Bran Fiber Show Distinct Metabolic Profiles in Rats as Investigated by a 1H NMR-Based Metabolomic Approach

This study aimed to examine the effect of pea fiber (PF) and wheat bran fiber (WF) supplementation in rat metabolism. Rats were assigned randomly to one of three dietary groups and were given a basal diet containing 15% PF, 15% WF, or no supplemental fiber. Urine and plasma samples were analyzed by NMR-based metabolomics. PF significantly increased the plasma levels of 3-hydroxybutyrate, and myo-inositol as well as the urine levels of alanine, hydroxyphenylacetate, phenylacetyglycine, and α-ketoglutarate. However, PF significantly decreased the plasma levels of isoleucine, leucine, lactate, and pyruvate as well as the urine levels of allantoin, bile acids, and trigonelline. WF significantly increased the plasma levels of acetone, isobutyrate, lactate, myo-inositol, and lipids as well as the urine levels of alanine, lactate, dimethylglycine, N-methylniconamide, and α-ketoglutarate. However, WF significantly decreased the plasma levels of amino acids, and glucose as well as the urine levels of acetate, allantoin, citrate, creatine, hippurate, hydroxyphenylacetate, and trigonelline. Results suggest that PF and WF exposure can promote antioxidant activity and can exhibit common systemic metabolic changes, including lipid metabolism, energy metabolism, glycogenolysis and glycolysis metabolism, protein biosynthesis, and gut microbiota metabolism. PF can also decrease bile acid metabolism. These findings indicate that different fiber diet may cause differences in the biofluid profile in rats.